Field of the Invention
The invention relates to a method for detecting the amplifications of sequences in the BRCA1 locus, which sequences have ends consisting of or are framed with sequence stretches present at least twice in the BRCA1 locus, and which amplification results in at least two or at least three, especially three, tandem copies of the amplified sequence. This invention also relates to methods for determining a predisposition to diseases or disorders associated with these amplifications, including predisposition to ovarian cancer or breast cancer. This invention also relates to a method for detecting amplifications with similar features in other loci.
Description of the Related Art
Breast cancer is the most common malignancy in women, affecting approximately 10% of the female population. Incidence rates are increasing annually and it is estimated that about 1.4 million women will be diagnosed with breast cancer annually worldwide and about 460,000 will die from the disease. Germline mutations in the hereditary breast and ovarian cancer susceptibility genes BRCA1 (MIM#113705) and BRCA2 (MIM#600185) are highly penetrant (King et al., 2003), (Nathanson et al., 2001). BRCA1 and BRCA2 genes, together with other genes such as NBR2 gene have been identified, characterized and mapped in the human genome and these data are publicly available. Screening is important for genetic counseling of individuals with a positive family history and for early diagnosis or prevention in mutation carriers. When a BRCA1 or BRCA2 mutation is identified, predictive testing is offered to all family members older than 18 years. If a woman tests negative, her risk becomes again the risk of the general population. If she tests positive, a personalized surveillance protocol is proposed: it includes mammographic screening from an early age, and possibly prophylactic surgery. Chemoprevention of breast cancer with anti-estrogens is also currently tested in clinical trial and may be prescribed in the future.
Most deleterious mutations consist of either small frameshifts (insertions or deletions) or point mutations that give rise to premature stop codons, missense mutations in conserved domains, or splice-site mutations resulting in aberrant transcript processing (Szabo et al., 2000). However, mutations also include more complex rearrangements, including deletions and duplications of large genomic regions that escape detection by traditional PCR-based mutation screening combined with DNA sequencing (Mazoyer, 2005). Only one amplification involving more than two copies has been reported so far (Hogevorst et al., 2003). This amplification is a triplication in the 3′ portion of the BRCA1 gene, involving exons 17-19 and caused by Alu recombination.
Techniques capable of detecting these complex rearrangements include Southern blot analysis combined with long-range PCR or the protein truncation test (PTT), quantitative multiplex PCR of short fluorescent fragments (QMPSF) (Hofmann et al., 2002), real-time PCR, fluorescent DNA microarray assays, multiplex ligation-dependent probe amplification (MLPA)(Casilli et al., 2002), (Hofmann et al., 2002) and high-resolution oligonucleotide array comparative genomic hybridization (aCGH) (Rouleau et al., 2007), (Staaf et al., 2008). New approaches that provide both prescreening and quantitative information, such as qPCR-HRM and EMMA, have recently been developed and genomic capture combined with massively parallel sequencing has been proposed for simultaneous detection of small mutations and large rearrangements affecting 21 genes involved in breast and ovarian cancer (Walsh et al., 2010). Other techniques described for the detection of these complex gene rearrangements include Molecular Combing (Herrick and Bensimon, 2009); (Schurra and Bensimon, 2009); (Gad et al., 2001), (Gad et al., 2002a), (Gad et al., 2003); (Cheeseman et al. 2012); (U.S. 61/553,906).
Prior art methods are unable to detect and/or characterize amplifications when such amplifications involve more than one additional copy of the amplified sequence and/or when the amplified sequence includes portions of sequence present in multiple copies in the wild-type BRCA1 gene or surrounding locus and/or when the amplified sequence belongs to a portion of the BRCA1 locus with very high repeat content. Here, the inventors provide methods to detect and/or characterize such amplifications and to detect and/or characterize amplifications sharing similar features in other genomic loci.